DNA Barcodes 2015; 3: 144–169

Review Open Access

Nicolas Hubert*, Kadarusman, Arif Wibowo, Frédéric Busson, Domenico Caruso, Sri Sulandari, Nuna Nafiqoh, Laurent Pouyaud, Lukas Rüber, Jean-Christophe Avarre, Fabian Herder, Robert Hanner, Philippe Keith, Renny K. Hadiaty DNA Barcoding Indonesian freshwater : challenges and prospects

DOI 10.1515/dna-2015-0018 the last decades is posing serious threats to Indonesian Received December 12, 2014; accepted September 29, 2015 biodiversity. , however, is one of the major sources of export for the international ornamental trade Abstract: With 1172 native , the Indonesian and home of several species of high value in . ichthyofauna is among the world’s most speciose. Despite The development of new tools for species identification that the inventory of the Indonesian ichthyofauna started is urgently needed to improve the sustainability of the during the eighteen century, the numerous species exploitation of the Indonesian ichthyofauna. With the descriptions during the last decades highlight that the aim to build comprehensive DNA barcode libraries, the taxonomic knowledge is still fragmentary. Meanwhile, co-authors have started a collective effort to DNA barcode the fast increase of anthropogenic perturbations during all Indonesian freshwater fishes. The aims of this review are: (1) to produce an overview of the ichthyological *Corresponding author: Nicolas Hubert, Institut de Recherche pour le researches conducted so far in Indonesia, (2) to present Développement (IRD), UMR226 ISE-M, Bât. 22 - CC065, Place Eugène an updated checklist of the freshwater fishes reported Bataillon, 34095 Montpellier cedex 5, France, E-mail: nicolas.hubert@ to date from Indonesia’s inland waters, (3) to highlight ird.fr the challenges associated with its conservation and Domenico Caruso, Laurent Pouyaud, Jean-Christophe Avarre, Institut de Recherche pour le Développement (IRD), UMR226 ISE-M, management, (4) to present the benefits of developing Bât. 22 - CC065, Place Eugène Bataillon, 34095 Montpellier cedex 5, comprehensive DNA barcode reference libraries for the France conservation of the Indonesian ichthyofauna. Nicolas Hubert, Sri Sulandari, Renny K. Hadiaty, Museum Zoolo- gicum Bogoriense (MZB), Division of Zoology, Research Center for Keywords: DNA barcoding;Checklist; Biology, Indonesian Institute of Sciences (LIPI), Jl. Raya Bogor Km46, Cibinong 16911, Java Barat, Indonesia. Kadarusman, Akademi Perikanan Sorong (APSOR), Kementerian Kelautan dan Perikanan, Jl. Kapitan Pattimura, Tanjung Kasuari, Sorong 98401, Papua Barat, Indonesia. 1 Introduction Arif Wibowo, Research Institute of Inland Fisheries, Agency for Marine and Fisheries Research – Ministry for Marine and Fisheries Biodiversity is not evenly distributed and aggregates Affair, Jl. Beringin No. 308, Mariana, Palembang 30763, Sumatera in restricted areas, some of which are currently facing Selatan, Indonesia. massive loss and as such, have been identified Frédéric Busson, Philippe Keith, Muséum National d’Histoire Natu- as biodiversity hotspots [1]. Among the 26 biodiversity relle (MNHN), UMR 7208 BOREA (MNHN-CNRS-UPMC-IRD), CP 026, 57 rue Cuvier, 75231 Paris Cedex 05, France. hotspots identified worldwide by Myers and colleagues Domenico Caruso, Nuna Nafiqoh, Jean-Christophe Avarre, Research [1], four are found in Southeast Asia (SEA) including and Development Institute for Health Control, Indonesian Indo-Burma (, , Laos, and Agency for Marine & Fisheries Research and Development, Jalan ), Sundaland (, Indonesia), Wallacea perikanan 12A, Depok, Java Barat, Indonesia (Indonesia) and Philippines hotspots. The exceptional Lukas Rüber, Naturhistorisches Museum der Burgergemeinde Bern, Bernastrasse 15, Bern 3005, Switzerland. concentration of biodiversity hotspots in SEA ranks Fabian Herder, Zoologisches Forschungsmuseum Alexander Koenig the region as one of the most diverse together with the (ZFMK), Leibniz-Institut für Biodiversität der Tiere, Adenauerallee Amazon and Congo River watersheds but threat levels 160, 53113 Bonn, Germany. actually rank them as the most endangered hotspots to Robert Hanner, Biodiversity Institute of Ontario and Department of date [2,3]. Among the four biodiversity hotspots identified Integrative Biology, University of Guelph, Guelph, ON, Canada in SEA, the two Indonesian hotspots are currently the most

© 2015 Nicolas Hubert et al. licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Unauthenticated Download Date | 12/4/15 10:41 PM DNA Barcoding Indonesian freshwater fishes: challenges and prospects 145 threatened and Sundaland in particular, is the one that nearly 1200 species of freshwater fishes have been either experienced the fastest increase of threat levels during described or reported from Indonesian inland waters and the last decade [3]. Including and the the rate of species discovery is still high as several tenth islands of , Java and (Fig. 1), this hotspot of species have been described from Indonesia during the exhibits one of the highest species richness and endemism last years [10-12]. The inventory of Indonesian freshwater for in SEA [2] and freshwater fishes are no fishes has been challenged since its earliest developments exception. For instance, among the 1200 species described by several limitations: (1) the Indonesian archipelago in Indonesia, nearly 900 species are observed in the hosts nearly 17,000 islands and most of them are remote Sundaland hotspot – c.a. 400 endemics – and constitute islands with limited access, (2) due to a complex political an important source of incomes from the international history, tracing the type specimens has been sometimes trade of ornamental fishes. The impoverishment of the challenging, particularly for the species described before ichthyodiversity in the Indonesian hotspots is of great the 1950’s [11], (3) the Indonesian ichthyofauna hosts concern, however, the taxonomic knowledge is still several large radiation of morphologically similar species incomplete and scattered in the scientific literature, what that have been subject to either multiple descriptions, arguably bridles the establishment of sounds conservation recurrent systematic revisions or overlooked diversity [11]. plans. Filling this gap is currently jeopardized by the fast Recently, the use of standardized molecular approaches degradation of the Indonesian natural due to a in some remote rivers in Indonesia emphasized that the large array of perturbations including mining, logging sole use of morphology in was limiting the activities, land burning for crop cultivation, deforestation estimation of species richness in some cases, as observed for land conversion (e.g. palm plantations) and water in Papuan rainbowfishes, for instance [13]. contamination [4, 5]. Freshwater fishes are particularly DNA barcoding is a system designed to provide at risk in Indonesia as their persistence is currently accurate, fast and automatable species identification by jeopardized by the interactions between ecological and using short and standardized gene regions as internal biotic (e.g. inland fisheries, introduction of alien species) species tags [14]. Initially proposed to circumvent the lack perturbations resulting in the modification of habitats, of taxonomists and available tools for species identification destruction of spawning grounds and the decline of [15-17], DNA barcoding has also been foreseen by several populations [6-9]. authors as a solution to speed up the pace of species The inventory of the Indonesian ichthyodiversity is discovery and open new perspectives in conservation [18- ongoing since the second half of the 18th century. So far, 20]. Given their high diversity and dramatic phenotypic

Figure 1. Map of Indonesia including the 23 islands considered in the present review (Appendix) with biogeographic provinces and their boundaries. 1, Bali; 2, Bangka; 3, Batam and Bintan; 4, Belitong; 5, Buru; 6, Java; 7, ; 8, Madura; 9 Natuna and ; 10, Suma- tera; 11, Bacan; 12, Celebes; 13, Ceram; 14, Flores; 15, Halmahera; 16, Indonesian Timor; 17, Lombok; 18, Sumba; 19, Sumbawa; 20, Ternate; 21, Talaud; 22, Aru; 23, Indonesia New Guinea.

Unauthenticated Download Date | 12/4/15 10:41 PM 146 N. Hubert, et al. changes during ontogeny, fish identification is not an easy To date, 1218 species belonging to 84 families have task. After almost a decade, it has become evident that been reported from Indonesian freshwaters including 1172 DNA barcoding presents several advantages compared native species from 79 families among which 630 species to morphological characters for species identification are endemic of the country (Table 1, Appendix). Amongst including: (1) intraspecific phenotypic variation often the 1218 species, 28 are exotic species corresponding to: overlaps that of sister taxa in nature, which can lead to (1) introduced species (i.e. 21 species from 7 families), incorrect identifications or species delineations [21,22], among which several belong to exotic families for (2) DNA barcodes are effective whatever the life stages Indonesian waters (e.g. Cichlidae, Loricariidae, under scrutiny [23,24] or available biological materials Peociliidae, Serrasalmidae), (2) imported species that for identification [25, 26], (3) spectacular levels of cryptic have not established yet (i.e. 7 species from 5 families), diversity have been frequently reported using DNA among which several species belong to 3 exotic families barcoding [21, 27-31]. (Poecilidae, Cichlidae, Salmonidae). Finally, 18 species An Indonesian initiative to DNA barcode all freshwater are cited from Indonesian waters but their presence is fishes is currently handled by the co-authors with the aim questionable and is likely to result from misidentifications. to assemble comprehensive DNA barcode libraries for With a density of 0.6 species per 1000 km2 (Table the Indonesian ichthyofauna. In this context, the present 1), Indonesia hosts one of the world’s highest density of review aims at exploring the potential contribution of fish species ahead of Brazil (0.37 species per 1000km2) DNA barcoding for the conservation of the Indonesian and the Democratic Republic of Congo (0.48 species ichthyodiversity. First, we provide updated statistics on per 1000km2), two countries known to host some the the Indonesian ichtyodiversity that we compiled from world largest and speciose tropical rivers [12]. The main several sources [10-12]. Second, we produce an overview of reasons for this exceptional diversity are the important the ichthyological exploration of Indonesian freshwaters fragmentation of the rivers across the numerous islands and highlight the major challenges is facing of the archipelago, together with the occurrence of in the country. To conclude, we discuss the prospects and several major biogeographical boundaries in the country challenges associated to the assembly of comprehensive including the Wallace and Lydekker lines (Fig. 1) and DNA barcode libraries for the Indonesian freshwater fishes. complex palaeoecological and geological histories [33-37]. The existence of biogeographic provinces in the Indonesian fauna is known since the seminal 2 Indonesian freshwater fishes: study of Wallace [38] and later by Lydekker in 1895, general considerations Weber [39] and Mayr [40]. Two major biogeographical boundaries are still recognized today in Indonesia (Fig. 1) The statistics presented here have been compiled from including the Wallace’s line separating Sundaland from Fishbase [12] and the Eschmeyer’s [32] as Wallacea and the Lydekker’s line separating Wallacea available on September 2014. An updated checklist of the SEA from Sahul [33]. Yet, two biodiversity hotspots have been freshwater fishes has been recently published by Kottelat [11] described by Myers and colleagues [1] that match those but this updated list was not incorporated in and biogeographical boundaries (Sundaland and Wallacea). Eschemeyer at the time we compiled the present statistics The distribution of species richness varies among those and connections with known species range distribution was three regions and Sundaland is the most speciose, both in not available at that time. As our objective is to provide an terms of absolute species richness and number of endemic updated assessment of the distribution of the Indonesian species with 899 and 431 species, respectively (Table 1). ichthyodiversity, we opted for the conservative list of species Sundaland also hosts the highest density of species with name in Fishbase. In this context, the species list provided 0.8 species and 0.38 endemic species per 1000km2. The here (Appendix) is readily available for the DNA barcoding endemism, however, is higher in Sahul with nearly 50% campaign of Indonesian fishes. Species range distributions of endemism while hosting only 20 percent of all the were compiled from Fishbase species page whenever they Indonesian endemic species. were detailed at the island level (Fishbase1 in the Appendix) The family is the most speciose family of or based on Fishbase Aquamap using valid records only the archipelago with 241 species followed by the families (Fishbase2 in the Appendix). Occurrences were refined or with 122 species, Osphronemidae with 81 species invalidated using Eschmeyer [32] and Kottelat’s checklists and with 60 species (Fig. 2A). The dominant [10,11] whenever additional or contradictory occurrences families, however, varies among biogeographical provinces where found. as Sundaland is dominated by the Cyprinidae (231 species;

Unauthenticated Download Date | 12/4/15 10:41 PM DNA Barcoding Indonesian freshwater fishes: challenges and prospects 147 ) 2 0.90 0.88 0.88 0.83 0.00 0.26 0.54 0.19 2.34 0.38 0.38 0.00 0.40 0.23 0.21 0.28 0.13 0.74 0.00 0.13 0.00 0.78 0.30 0.12 0.29 0.29 0.35 Density Density (Sp/1000km 13.16 28.57 7.69 22.22 0.00 15.49 45.51 8.33 42.11 35.22 47.94 0.00 47.26 7.55 12.00 9.80 8.70 20.00 0.00 11.11 0.00 100.00 44.57 4.55 49.80 49.02 Percent (total (total Percent species) all 0.01 0.02 0.00 0.01 0.00 0.05 0.46 0.00 0.01 0.25 0.68 0.00 0.11 0.01 0.00 0.01 0.00 0.01 0.00 0.00 0.00 0.00 0.13 0.00 0.19 0.20 Percent (total (total Percent endemics) Endemics 5 10 2 4 0 33 294 1 8 162 431 0 69 4 3 5 2 4 0 2 0 1 82 1 124 125 630 of species N. of ) 2 Density Density (Sp/1000km 6.80 3.10 11.40 3.80 1.70 1.70 1.20 2.30 5.60 1.10 0.80 4.40 0.80 3.00 1.70 2.90 1.50 3.70 0.80 1.20 169.20 0.80 0.70 2.60 0.60 0.60 0.6 Percent (total (total Percent species) all 0.30 0.90 0.10 0.20 0.20 13.30 35.30 0.30 0.10 23.50 74.00 0.10 6.70 1.70 0.20 0.40 0.30 0.20 0.20 0.10 0.10 0.10 15.00 0.50 15.40 16.00 All species species N. of 38 35 26 18 16 213 646 12 19 460 899 8 146 53 25 51 23 20 9 18 11 1 184 22 249 255 1172 of family N. of 14 23 18 14 14 54 66 11 8 64 73 6 31 25 13 23 13 10 5 10 9 1 37 8 45 45 79 Surface (km2) Surface 5561 11330 2280 4800 9505 126700 539500 5290 3420 425000 1133386 1800 174600 17418 14300 17780 15770 5435 11153 15448 65 1285 275054 8563 421981 430544 1838984 Island Bali Bangka Batam/Bintan Belitung Buru Java Kalimantan Madura Natuna/Riau Sumatera Total Bacan Celebes Seram Flores Halmahera Indonesian Timor Lombok Sumba Sumbawa Ternate Talaud Total Aru Indonesian New Guinea Total Biogeographic Biogeographic domain Sunda Wallacea Sahul Code Map Code Summary statistics of the Indonesian ichthyofauna including surface of islands, species richness, endemism and species density for the 23 major islands considered in the present in the present considered islands the 23 major for density species and endemism richness, species islands, of surface including ichthyofauna the Indonesian of statistics Summary 1. Table review. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Total

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Fig. 2B) while Wallacea and Sahul are dominated by the Wallacea and Sahul, (2) the second since 1978 until now Gobiidae (52 species) and Melanotaeniidae (47 species), corresponding to the modern ichthyological exploration respectively (Fig. 2C & 2D). A similar pattern is observed of the Indonesian freshwaters and the development of when considering endemism, as the family Cyprinidae comprehensive systematic studies of SEA freshwater is also the richest family with 132 endemic species, all fishes. Many species in Sahul have been described during observed in Sundaland (Fig. 3A & 3B). Endemism in the the modern period (e.g. Melanotaenia spp.; Fig. 5) and family Osphronemidae is also among the highest with are to be attributed to the development of transportation 59 endemic species, all observed in Sundaland. The systems and eased access in the eastern part of Indonesia Melanotaeniidae is the third family with 45 endemic (e.g. Indonesian Papua). Many species, however, have species and exhibits the highest level of endemism, as 45 been described from Sundaland during the same period of the 47 species are endemic of Sahul. The family ranking and correspond to the reappraisal of diversity in complex according to the number of endemic species follows the groups of closely related species (e.g. spp., Betta same pattern as for the whole ichthyofauna in Wallacea spp., Parosphronemus spp., Clarias spp., Ompok spp., with Gobiidae, Adrianichthyidae and Telmatherinidae Osteochilus spp.; Fig. 5) as a consequence of an increased being the richest families with 23, 18 and 17 endemic taxonomic and systematic knowledge of the Indonesian species, respectively. The same pattern applies in Sahul ichthyofauna (e.g. Rasbora) as well as the use of integrative with Melanotaeniidae, Eleotridae and Gobiidae being taxonomy including molecular (e.g. Clarias, the most diverse with 45, 20 and 14 endemic species, Pangasius). The impact of the modern ichtyological respectively. exploration of Indonesian waters is also reflected by the The earliest descriptions of SEA native species were distribution of the number of description of endemic done in 1758 by Linnaeus (e.g. Clarias batrachus). The species per decades as 295 of the 631 endemic species rate of species description in SEA has been low until have been described since 1978, that is 47 percent of the the 1850’s with 204 species described between 1848 and Indonesian endemic species (Fig. 4B). 1857 (Fig. 4A). Since then, the rate of species description The majority of the species described or recorded per decade has undergone two additional peaks: (1) in Indonesia are less than 15 cm with 238 species the first between 1898 and 1917 (i.e. development of the of less than 5 cm, 322 between 5 and 10 cm and 166 Dutch colonies in SEA) with numerous description from between 10 and 15 cm (Fig. 4C; Fig. 5). Most of the

Figure 2. Ranking of the families according to their species richness (Appendix). A, Indonesia; B, Sundaland; C, Wallacea; D, Sahul.

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Figure 3. Ranking of the freshwater fish families according to their number of endemic species (Appendix). A, Indonesia; B, Sundaland; C, Wallacea; D, Sahul.

Figure 4. Number of species description per decade and distribution of maximum length classes for the native freshwater fish species of Indonesia. A, all species; B, endemic species; C, distribution of maximal length per 5 cm classes.

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Figure 5. Selected photographs of some emblematic species and lineages of the Indonesian ichtyofauna. 1, sp. (Picture A. Wibowo); 2, Parachelo oxygastroides (Picture N. Hubert); 3, Rasbora patrickyapi (Picture L. Rüber); 4, Desmopuntius rhomboocellatus (Picture L. Rüber); 5, cf. carbunculus (Picture L. Rüber); 6, trichopterus (Picture N. Hubert); 7, Parosphromenus opallios (Picture L. Rüber); 8, Trichopsis vittata (Picture N. Hubert); 9, Betta hendra (Picture L. Rüber); 10, Anabas testudineus (Picture N. Hubert); 11, Nemachei- lus fasciatus (Picture N. Hubert); 12, Pangio pulla (Picture L. Rüber); 13, Lepidocephalichthys hasselti (Picture N. Hubert); 14, Parakysis notialis (Picture ); 15, Glyptothorax platypogon (Picture N. Hubert); 16, Clarias batrachus (Picture N. Hubert); 17, Hemibagrus nemurus (Picture N. Hubert); 18, Pangasianodon hypophthalmus (Picture N. Hubert); 19, Channa striata (Picture N. Hubert); 20, lopis (Picture A. Wibowo); 21, Macrognathus aculeatus (Picture N. Hubert); 22, Belobranchus segura (Picture N. Hubert); 23, Butis amboinensis (Picture N. Hubert); 24, Sicyopus zosterophorum (Picture N. Hubert); 25, Lentipes mekongaensis (Picture R. Hadiaty); 26, Sicyopterus lagocephalus (Picture N. Hubert); 27, Mugilogobius hitam (Picture F. Herder); 28, Hemirhamphodon pogonognathus (Picture L. Rüber); 29, Nomorham- phus rex (Picture F. Herder); 30, Oryzias eversi (Picture F. Herder); 31, Melanotaenia fascinensis (Picture L. Pouyaud); 32, Telmatherina antoniae (Picture F. Herder).

Unauthenticated Download Date | 12/4/15 10:41 PM DNA Barcoding Indonesian freshwater fishes: challenges and prospects 151 descriptions during the modern period correspond Recent molecular phylogenetic studies have provided to small sized species, what highlight the difficulties important insight into intra- and interrelationships associated with the characterization and delineation of the most speciose families. With more than 3200 of lineages for those small sized species [e.g. 13,41-45]. species, the are the most species rich The distribution of the number of species description lineage. The last few years have seen an increase in per decade highlights that taxonomic studies have been molecular phylogenetic studies addressing Cypriniform fragmented during the pre-modern period but also intrarelationships [e.g. 47] or focusing on different that despite two centuries and a half of ichthyological Cypriniform subgroups. Cypriniformes is divided into inventories; many species are still described regularly the two superfamilies Cobitoidea and Cyprinoidea throughout Indonesia, not only from the most remote and a total of families ranging from 11 [12] to 13 [47] rivers and lakes of Wallacea and Sahul. depending on the authors. The Cobitoidea comprise the families Catostomidae, Gyrinocheilidae, Vaillantellidae, , Ellopostomatidae, Barbuccidae, Balitoridae, 3 Integrative taxonomy in Indone- Serpenticobitidae, Nemacheilidae (Fig. 5). With the sian fishes: species discovery and exception of the Catostomidae and the Serpenticobitidae, all these families are found in Sundaland (Appendix). systematic knowledge On the other hand the Cyprinoidea include the families Psylorhynchidae and Cyprinidae, the later one is 3.1 The Sundaland ichthyofauna ubiquitous in Sundaland. Several attempts to subdivide the species rich family Cyprinidae by erecting new families Sundaland is the western most biogeographic domain of the have been done recently [e.g.` 48,49] but these systematic archipelago and also the most speciose either considering rearrangements based on molecular phylogenetic studies the number of widespread or endemic species (Table have not been adopted by subsequent workers [e.g. 11,50]. 1). Sundaland is also the only domain where families of Recent work on Cobitoidea intrarelationships [e.g. primary freshwater fishes are dominant in term of species 51,52] mainly focused on the phylogenetic placement richness (e.g. Cyprinidae, Osphronemidae, Bagridae, of several rare and species-poor lineages such as the Balitoridae, Siluridae, Cobitidae; Fig. 5). This relative Barbuccidae (one , two species, one of which is importance is largely determined by the palaeoecological found in Borneo), Ellopostomatidae (one genus with two history of Sundaland and past connections with centers of species, one of which is found in Borneo), Vaillantellidae diversification and refuges for primary freshwater fishes (one genus with three species, all of which are found (e.g. Cypriniformes, Siluriformes; Fig. 5). For instance, in Borneo), and Gyrinocheilidae (one genus with three Kalimantan has played the role of a center of diversification species; one species Gyrinocheilus pustulosus occurs in for several lineages of primary freshwater fishes such as Borneo). Cobitidae intrarelationships were addressed a the Balitoridae (e.g. Gastromyzon, Homaloptera), Cobitidae few years ago by Slechtova and colleagues [53,54] who (e.g. Pangio), Cyprinidae (e.g. Eirmotus, Lobocheilos, explored the genera relationships inside the family Osteochilus, Desmopuntius, Rasbora), Osphronemidae (e.g. including several well-known lineages from Sundaland Betta, Parosphronemus), Akysidae (e.g. Acrochordonichthys, such as Acanthopsis, Kottelatlimia, Lepidocephalichthys, Parakysis, Pseudobagarius), Bagridae (e.g. Pseudomystus) Pangio or the monotypic genus Chromobotia who’s sole and Sisoridae (Glyptothorax) fostering the settlement of representative the clown (C. macracanthus) from high level of endemism for those groups. De Bruyn and Borneo and Sumatra is an important ornamental fish colleagues [35], for instance, have demonstrated that species. The largest genus within the southern lineages of Borneo’s diversity (including Kalimantan in Indonesia and Cobitidae is Pangio with currently 32 recognized species Sabah and provinces in Malaysia) resulted not including several well-known and valued ornamental only from the accumulation of immigrants but also from species, such as the kuhli . Bohlen and colleagues in situ diversification that further enabled the dispersal of [55] explored recently the phylogenetic relationships species to other islands in Sundaland including Java and among Pangio species and suggested the presence of Sumatra. Dispersal among Sundaland islands has been several cryptic species within the genus. made possible by eustatic changes during glacial times The phylogenetic intra- and interrelationships of through the Pleistocene that created connections between the family Cyprinidae have been certainly the most the islands of Sundaland but also with the continent studied, with several studies looking at the [9,10,34,46]. of the entire family or focusing on a single subfamily

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[43,56-62]. The Cyprinidae subfamily Danioninae, a large range from free-spawning tactics without parental care group composed predominantly of South and Southeast to substrate spawning, submerged plant nest building, Asian , is among the best known subfamily bubble nesting, and mouth-brooding with parental care. and harbors several genera that are very popular in the While the most comprehensive molecular phylogenetic ornamental fish trade (e.g. Boraras, Danio, Rasbora, framework for labyrinth fishes has been provided by Trigonostigma, ). Their intra-relationships Rüber and colleagues [65], molecular studies focusing on have also been largely explored recently [43,62-64]. This diversity patterns at the genus level or phylogeographic subfamily harbors many miniature taxa including highly studies are still scarce for this group [but see 66]. developmentally truncated ones [62], some of which Recent studies on the phylogeography of Sundaland have only been described very recently. For example, the fishes based on mitochondrial genes confirmed that the genus Paedocypris that includes the world’s smallest fish evolutionary history of Sundaland fishes was marked by species (), was only described in the landmass dynamics during the Pleistocene resulting 2006 [42]. Paedocypris currently includes three endemic from the eustatic fluctuations. Nguyen and colleagues [67], species of the highly stenotopic found for example, have highlighted that species of the cyprinid in Peninsular Malaysia, Sumatra and Borneo. The genus genus Tor widely distributed in SEA, showed contrasting Fangfangia, also a peat swamp forest endemic, contains patterns of diversity depending on the proximity to only one species (Fangfangia spinocleitralis) that is found Sundaland as species widely distributed in Sundaland in Central Kalimantan, Borneo [44]. Miniaturized taxa are exhibits high levels of cryptic diversity and sister-lineages often difficult to place phylogenetically - and Paedocypris with trans-island relationships. In Tor douronensis, in is no exception - due to the frequently observed trend particular, they observed four mitochondrial lineages towards reduction and simplification of various with allopatric distributions across Peninsular Malaysia, structures and organs [62]. The phylogenetic placement Borneo and Sumatra. Along the same line, De Bruyn and of Paedocypris in the family Cyprinidae has been subject colleagues [34] evidenced that species boundaries in the to intense debate during the last years [e.g. 49,50,62-64] genus Dermogenys, and Hemirhamphodon and results highlighted that more molecular phylogenetic derived from mitochondrial sequences were matching studies are needed to provide a robust phylogenetic palaeodrainage boundaries during periods of low sea framework to better understand Cyprinidae diversity. levels but poorly matched current islands boundaries. Labyrinth fishes or constitute another The authors also detected cryptic diversity for widespread major component of the Sundaland ichthyofauna. They species with insular endemic mitochondrial lineages. The exhibit an Afro-Asian distribution and are arranged study by Pouyaud and colleagues [68] on the genus into three families: Osphronemidae (over 120 species Clarias yielded similar results as an important level of in three subfamilies Osphroneminae, Macropodisnae, cryptic diversity was detected for the widespread Clarias and ), Helostomatidae (one species, species in Sundaland (e.g. C. olivaceus, C. meladerma, C. Kissing ), and Anabantidae (around 30 species). punctatus and C. nieuhofii) with range distribution of the While the former two families are restricted to Asia, the mitochondrial lineages rather matching palaeodrainages Anabantidae are found in both Africa and Asia with the than modern islands boundaries. widespread Anabas testudineus, the climbing perch being the sole Asian member of this family. Although, 3.2 The Wallacea ichthyofauna a comparatively small group, Anabantidae exhibit a striking variation in size, ranging from dwarfed forms Located east of Wallace’s line, ’s freshwater such as Parosphromenus ornaticauda, with 19 mm fauna sharply differs from that of Sundaland, including standard length, to large forms such as the giant goramies the nearby Borneo. Besides of euryhyaline lineages, or of the genus Osphronemus, with up to 70 cm standard such with larval stages drifting across brackish or marine length. A number of species play an important role as food barriers, Sulawesi’s inland waters are well known for fishes and are widely used in aquaculture (Osphronemus, their landlocked radiations of invertebrates and fishes Anabas, Helostoma; see elsewhere in the text), whereas [reviewed in 69]. The most spectacular and diverse of others are important and highly colorful fishes these radiations occurs in the ancient lakes of Central such as the fighting fishes (Betta spp.), and the licorice Sulawesi, namely the Malili Lakes system, and Lake Poso. (Parosphromenus spp.). Labyrinth fishes show Besides of lacustrine radiations of gastropods, crabs, an astonishing diversity in breeding behavior that is , and other invertebrates, the ecosystems of these rarely found in any other fish group. Reproductive modes isolated, deep, and long-term stable freshwater lakes are

Unauthenticated Download Date | 12/4/15 10:41 PM DNA Barcoding Indonesian freshwater fishes: challenges and prospects 153 characterized by fish species flocks. The most spectacular 16 percent to the Indonesian ichthyodiversity and 20 radiation in Sulawesi is observed in the sailfin silversides percents to the total number of endemic species in the (Telmatherinidae; Fig. 5) from the interconnected Malili country, some lineages (e.g. Melanotaenia, Mogurnda) Lakes, including the 590 m deep graben-lake Matano exhibit up to 100 percent of endemism (Table 1). The [41,70-72]. These small, Atheriniformes fishes with colorful rainbowfishes belonging to the genus Melanotaenia male ornamentation are closely related to New Guinea’s (Fig. 5) represent the most speciose group of freshwater and ’s rainbowfishes, and serve as model system fishes in Indonesian Papua with 33 species that are for the study of speciation mechanisms [summarized all endemic to the region. Inside Indonesian Papua in 73], and the maintenance of color polymorphisms in in particular, Allen [86] initially suggested that the nature [e.g. 74,75]. western part of the island, also known as the Bird’s Mitochondrial sequencing was performed as one of the Head peninsula, might be a hotspot of rainbowfish initial steps for estimating sailfin silverside species diversity diversity according to the initial exploration of the [41,76]. In agreement with morphology and nuclear data, area that led to the description of several new species. mitochondrial markers resolved major lineages of the The subsequent ichthyological exploration of the area radiation [41], and provided valuable insights into the confirmed this hypothesis through the description of phylogenetic history, the origin, and time estimates of its several additional species of Melanotaenia based on divergence [77]. Mitochondrial sequences, however, are less a screening of morphological characters [87-89] and suited for discriminating morphospecies within the major leading the citation of 13 species from the Bird’s Head. lineages of this radiation [41,72]. For instance, introgressive The large-scale and standardized sequencing of the COI hybridization between stream- and lake-dwelling sailfin gene for rainbowfish species from the genus Melanotaenia silversides [78] has lead to severe confusion in previous with the aim to re-estimate the diversity in Indonesian Papua studies relying solely on mitochondrial markers for species provided an unprecedented level of species discovery in the delineation [e.g. 73,76,79]. group [13]. Kadarusman and colleagues [13] have detected The absence of species-level resolution in mitochondrial 30 mitochondrial lineages among the 13 nominal species markers is not unexpected in evolving radiations, of Melanotaenia in the Bird’s head, all being distributed in particularly when ecological speciation takes place in allopatry and restricted to a single watershed. This cryptic the absence of geographical barriers, as observed in diversity has been further confirmed by the high levels of the endemic lacustrine species of Lake Matano [80,81]. genetic differentiation based on microsatellite genotyping Introgressive hybridization among closely related species and the discovery of several diagnostic morphological is also known to blur phylogenetic signal, particularly characters [90]. By doubling the number of known species for maternally inherited mitochondrial markers. in the area, Kadarusman and colleagues [13] and Nugraha Mitochondrial-based studies of species diversity in lake and colleagues [90] highlighted that the diversity of the ecosystems, however, provided valuable information. In family was largely underestimated and pinpointed the some of the invertebrate radiations of the Sulawesi Lakes, urgent need to re-appraise the diversity of this group in such as Atyiidae shrimps, or Geocarcinucidae crabs, other region of Indonesian Papua for national conservation mitochondrial sequences have been generally very well plans. suited for discriminating species. The use of mitochondrial DNA sequencing lead to In the case of Sulawesi’s ricefish radiation, which spectacular levels of species discovery in Indonesian comprises riverine as well as lacustrine species (Fig. 5), Papua because allopatric speciation in fragmented mitochondrial sequences contributed significantly to landscapes has been the major driver of the rainbowfish species discovery and improved our current understanding diversification [13]. Hybridization is known to occur easily of their evolutionary history [82-85]. Nevertheless, a few among Melanotaenia species and inter-generic hybrids species flocks that evolved in lacustrine environment, as have even been observed [86], a result that was confirmed in L. Towuti for instance, display some intricate patterns although by Kadarusman and colleagues as introgressive of shared polymorphism [83]. hybridization between Melanotaenia and Glossolepis species was observed in one case. Phylogenetic inferences 3.3 The Sahul ichthyofauna based on mitochondrial DNA also helped estimating the time frame of the Melanotaenia diversification and Located east of Lyddeker’s line, the ichthyofauna confirmed the influence of the complex geological history of Sahul shares similar characteristics with the of the Papua island in shaping the current distribution of ichthyofauna of Wallacea. While contributing by only the family [13,91].

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Worth mentioning, a similar pattern of endemism the settlement of this gradient including the existence of is observed in the genus Mogurnda encompassing nine a center of overlap among distinct biogeographic domains species, all being endemic of Indonesian Papua. The [29,107,108], the highest connectivity of the Pleistocene genus Mogurnda is the sole genus of the family Eleotridae coral reef refuges in the Indo-Australian archipelago [109] to exhibit 100 percent of endemism. The descriptions of and geometric constraints on the distribution of species Mogurnda species have all been done during the 1990’s richness [110,111]. by Allen and colleagues [92-95] and a recent survey of With eight valid genera (Sicyopterus, Cotylopus, mitochondrial diversity in closely related Australian Smilosicyopus, Sicyopus, Akihito, Stiphodon, Lentipes Mogurnda species revealed that cryptic diversity was and Sicydium; Fig. 5) and over 110 species [112], the prevalent in the genus and the actual diversity to be as amphidromous fishes of the subfamily much as twice the extent number of nominal species (Teleostei: Gobioidei) constitute some of the major today [96]. contributors to the freshwater fish biomass in river systems of remote tropical islands [113, 114]. The first 3.4 Euryhaline and diadromous lineages molecular phylogeny of the Sicydiinae estimated that with regional distribution the group emerged quite recently around 6 – 12 Myrs ago [115] and the ancestral area reconstruction made by Euryhaline and amphidromous species represent an Taillebois and colleagues [112] suggests that Sicydiinae important contribution to the Indonesian ichthyofauna, might have emerged within the Indonesian shelf that has sometimes in a high proportion as in Wallacea and Sahul probably been a key area in the diversification of some (e.g. Gobiidae, Eleotridae, Ariidae, Apogonidae, Plotosidae, clades such as Sicyopus and the Cotylopus/Smilosicyopus Terapontidae, Ambassidae, Mugilidae, Anguillidae). group [116]. In Indonesia, nearly 11 type localities of This relative importance is largely determined by the Sicydiinae are found among the 26 species reported in palaeoecological history of each biogeographic domain Indonesia, that is 43% of the Sicydiinae species cited in in Indonesia and past connectivity (cf. section related to the country. The systematic of this group, however, is still Sundaland). Islands of Wallacea and Sahul have been largely controversial and calls for a finer assessment of isolated from regional pools of primary freshwater fishes the family diversity in Indonesia. The recent description by contrast with Sundaland that has been repeatedly of four new Lentipes and Sicyopus species confirms that connected to the SEA continent. Several insular the Sicydiinae fauna is still poorly known in Indonesia radiations happened in both Wallacea (e.g. Telmatherina, [117,118]. The molecular phylogeny of the group, however, Oryzias, Nomorhamphus) and Sahul (e.g. Melanotaenia), evidenced that molecular lineages generally match species however, several families including euryhaline and boundaries and confirmed that Sicydiinae inventories diadromous fishes (cf. example above) constitute an may benefit from a standardized sequencing for species important contribution to the diversity of both Wallacea delineation and assignment [115]. and Sahul. Past connectivity among islands, however, had different consequences for those lineages. This trend is particularly evident for amphidromous species 4 Molecular diagnostic, DNA with marine larval stages enabling connectivity among barcoding and management of ich- populations from different islands and exhibiting highest colonization potential due to their ability to achieve large- thyological resources scale dispersal as a consequence of long larval survival in oceans [97,98]. As a consequence, such amphidromous 4.1 Fisheries management lineages show more similarity in their population dynamic with coastal marine fishes whose adults are sedentary Most of the large rivers in Sundaland host inland fisheries and connectivity is maintained through larval dispersal as that supply freshwater fish markets mainly dedicated to observed in coral reef fishes [99-101]. Species richness in local consumption as in central Sumatra (e.g. ) Indo-Pacific reef fishes exhibits a marked heterogeneity [119] and west Kalimantan (e.g. ) [120]. Some following a gradient that peaks in the Philippines and fisheries are specialized on species with high economic Indonesia and decreases toward the west and the east and socio-cultural values such as the featherbacks (Chitala [102-106]. The origin of this gradient of diversity has spp.) or (Tor spp.) in Sundaland [119,120]. been intensively debated during the last two decades as The Chitala spp. belong to the family several mechanisms have been proposed to account for that exhibits only few species distributed mainly in SEA

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(i.e. 7 species, all cited from Indonesia) and 3 species in previously described based on mitochondrial sequences Africa [12]. Family’s age estimates based on complete [67,125]. mitochondrial genomes, however, date the origin of the Understanding migration of early life stages is family and the separation between African and Asian of utmost importance for fisheries management, a lineages during the early cretaceous around 160 – 180 knowledge that is critically needed when considering Myrs ago and around 130 – 150 Myrs ago, respectively amphidromous species including a marine larval stage [121]. Thus, aside of high socio-economical interest, such as the Sicydiinae (Gobiidae). Due to their marine featherback are also of high patrimonial importance larval stage (cf. previous section), they constitute the most in SEA. The giant featherback (Fig. 5) is very popular in important contributor to the biomass in river systems Sumatra owing to the large size it can achieve (i.e. 1.5 m) of remote tropical islands [113]. The biomass of larvae and its nutritional qualities [122] that rank it as one of the migrating upstream is so important during some periods most sought fish for consumption in the area [119] and the of the year that they represent an important source of most expansive fish species in the central Sumatra fish food for local human populations in some archipelagos markets [119,122]. As a consequence, the fishing pressure [127], particularly in the Philippines and Indonesia [128]. has grown dramatically during the last decades and The naturally unstable and ephemeral characteristics giant featherback fisheries have experience a stunning of these tropical systems, which have become even decline in productivity as exemplified in the Kampar more so in recent years as a result of anthropogenic river where the annual production dropped from 50.2 perturbations, however, make the Sicydiinae particularly tons in 2003 down to 7.6 tons in 2007 [123]. Sequencing of vulnerable [129]. Thus, harvesting this resource is highly mitochondrial genes, however, indicated that at least four unsustainable, on account of the complexity of the species distinct cryptic mitochondrial lineages may be observed in life cycle. For such species, estimating the larval biomass the populations of the giant featherback in Sumatra [119]. per species during those migrations is tightly linked to A distinct stock, for instance, has been evidenced from the the ability to identify larvae. Nevertheless, given their Kampar river and distribution of nucleotidic diversity at high diversity and dramatic phenotypic changes during mitochondrial sequences highlights that populations of development, fish larvae identification is not an easy task the giant featherback are highly structured spatially in and Sicydiinae and not an exception to this [24]. The first Sumatra suggesting that local scale is more appropriate molecular phylogeny of the group suggested that the use of for fisheries management than regional plans. mitochondrial sequences for species identification may be Another iconic group of species of the Sundaland possible since no mismatch between species boundaries inland fisheries is the mahseer (Tor spp.; Fig. 5), an and molecular lineages was detected [115]. economically important genus of Cyprinidae. Present from India to Indonesia and Malaysia, Tor spp. have played a 4.2 Ornamental trade great role in local fisheries in most of the countries where they occur [e.g. 120,124]. Tor spp. are amongst the most Over the last decades, the international trade in ornamental popular Indonesian freshwater fishes both for their table fishes has grown rapidly from small export fisheries in the and cultural values owing to their large size (i.e. 1 m) and Central Amazon and SEA to an industry that involves most nutritional qualities. The market price for Mahseer in tropical regions and generates some 200-300 millions Indonesia and Malaysia is amongst the highest ranging USD annually. The value of the ornamental fish exports from 16 to 60 US$ per kg [120], however, its abundance has been reported to increase by 14% per year between has dwindled substantially in their natural habitat since 1985 and 1996 [130,131]. The increase of the international the early 90’s [10,125]. In addition, most Tor spp. are trade of ornamental fishes over the years poses a great rheophilic species know to migrate downstream during challenge to national authorities in SEA since this market the onset of the raining seasons for spawning [12] and as is still depending on capture in the wild for many species such have been largely affected by the development of [132, 133]. In 2003, Indonesia ranked as the second highest dams. Furthermore, Mahseer’s controlled breeding is still exporting country of ornamental fishes by contributing to poorly developed mainly due to the difficulties to induce nearly 7% of the global trade [134]. maturation and ovulation by using hormonal treatments Blackwaters associated with peat swamp forests [126]. The dynamic of migration and life history traits are in Sundaland, for example, are home to many species still poorly known in Indonesian populations of interesting the international ornamental fish trade from Sumatra and Kalimantan, however, cryptic diversity including Betta spp., Parosphronemus spp., and population structure among rivers have been spp., Rasbora spp. and Boraras spp. (Fig. 5) among the most

Unauthenticated Download Date | 12/4/15 10:41 PM 156 N. Hubert, et al. prominent examples [135]. Blackwater swamps, however, regional main town of Cairns, it was recommended to are experiencing an alarming rate of land conversion. review the protective status for all Sicydiinae gobies of the Forest loss in the lowlands of Sumatra and Kalimantan, country as the interest in collecting Stiphodon species is the two Indonesian provinces containing the largest areas likely to be high for native fish hobbyists [141]. of peat swamp forest and accounting for more than 70% of Stiphodon spp. are easy to collect and are accessible at forest clearing in the country from 1990–2005, resulted in low altitude in small streams. As a consequence, the Opal a staggering 41% loss in total area in just 15 years [136,137]. cling goby, Stiphodon semoni, widespread in Indonesia, Thus, the need to improve our taxonomic knowledge on has recently been listed in Australia under the Environment either collected in the wild or reared Protection and Biodiversity Conservation Act. A recent and bred captive for ornamental purposes is crucial in paper reviewing the Stiphodon species in Sumatra, and Indonesia. describing one as new [143], used more than half of the Over the past few decades, the Asian specimens of all three Stiphodon species examined from formosus has acquired an emblematic ornamental traders in . Stiphodon gobies are status in most Asian countries as a very popular but often sold commercially as ornamental fish. These three extremely expensive aquarium fish, which has led to species (S. ornatus, S. semoni and S. maculidorsalis) are its overexploitation and its inclusion in the CITES list of common and found in many pet shops often selling them species threatened with [138]. In a phylogenetic on the Internet, even if the later is supposed to be endemic analysis based on the sequencing of the mitochondrial to Sumatra. The western slope of Sumatra is believed to be cytochrome b gene and supported by several diagnostic the main source of Stiphodon for the ornamental trade. To morphological characters, Pouyaud and colleagues [139] date, the large-scale captive breeding of Stiphodon species demonstrated that the four different color varieties of is still not tuned due to difficulty in feeding their small S. formosus were distinct species. Those included the larvae, due to their amphidromy and long pelagic larval “Silver” described as Scleropages macrocephalus, the duration [144]. Therefore, all Stiphodon species available “Golden” belonging to S. aureus, the “Red” identified as in the ornamental market are collected from the wild. S. legendrei, and the “Green” restricted to S. formosus. The same constraints apply to other Sicydiinae such as The authors also proposed to reconsider the status of S. Sicyopterus, Lentipes and Sicyopus that are occasionally aureus and S. legendrei as critically endangered species available in the ornamental market. because both species previously considered as color varieties of S. formosus were in fact exhibiting highly 4.3 Aquaculture confined geographic distribution very dependent on the nature and quality of the habitat (e.g. peatswamps), and Freshwater , which represents 60% of therefore threatened of extinction due to habitat loss aquaculture production in Indonesia, is an ancient and and overexploitation for ornamental purposes [139]. The traditional production that is essential for the country’s capture of specimen in the wild for the international food security. The Indonesian ichthyofauna has several ornamental fish trade also poses serious and immediate iconic species in the traditional fish farming and the giant threats to other Indonesian fishes not associated goramy (Osphronemus goramy) is one of the most popular with the peat swamps. The (Chromobotia species in Sundaland owing to its table values [10]. The macracanthus), for instance, is another prime example of giant goramy (Fig. 5) is an omnivorous-phytophagous an iconic species of the ornamental trade whose captive species fed in captivity with a variety of cultivated plants. breeding has been tuned only recently [140] and its Its traditional rearing may be considered as a prime marketing has been entirely sustained during decades example of integrated production system in agricultural from captures in the wild. landscapes as it generally occurs in ponds associated with The Gobiidae subfamily Sicydiinae also hosts several plantation of cassava and papaya whose leaves are used important species in the international ornamental trade. directly for feeding adults [145]. Some examples are given in Australia [141,142] where Java is the cradle of the traditional rearing of the giant five Stiphodon species were recently discovered in small goramy as Javanese and Sundanese peoples peddled the fast-flowing rivers. These species are vulnerable to over- rearing of this species throughout the history of their harvesting for the aquarium trade as a result of their settlements in Sundaland and later fostered the spread colorful appearance, particularly in male, and interesting of its aquaculture in the country. Huet [146], for instance, behaviors [113]. Given the small population size of these already mentioned the rearing of the giant goramy in species and the proximity of these populations to the Indonesia as one of the most important freshwater

Unauthenticated Download Date | 12/4/15 10:41 PM DNA Barcoding Indonesian freshwater fishes: challenges and prospects 157 aquaculture in the 50’s. More recently, considering the being exotic and the later being the most common in the high commercial value and growing market demand for country [156]. These species present distinct breeding this species, fish farmers have intensified the breeding performance (e.g. Legendre, 2008 #2779}, however, fertile over the past few years and new goramy’s farms have hybrids are easily obtained in rearing condition leading developed in several areas in Indonesia, particularly in to altered breeding performances and potential risks of central Java that is currently the main source of juveniles introgression of natural populations of the native species, in Sundaland. As a consequence, the giant goramy is P. djambal [157]. currently being translocated in many islands throughout the country. 4.4 Border biosecurity and invasive species The genus Osphonemus encompasses four nominal species among which, three are cited from Indonesia but Invasive species may pose serious threats to native only O. goramy has been reported from Java, Sumatra freshwater faunas, especially in insular, isolated and Kalimantan (Table 1). Owing to its early history of ecosystems. There are numerous freshwater fishes that are domestication in Sundaland, however, nearly thirteen regularly transplanted to areas outside of their original cultivated strains are currently recognized in the Javanese ranges as a consequence of increased human activities cultivated O. goramy, several of them exhibiting distinctive [158, 159]. Invasive species have not only fostered coloration patterns and life history traits including the disruption of global patterns in freshwater fish fecundity, mortality and growth rates (D. Caruso, pers. communities [160], but some of these have turned out as obs.). Setijaningsih and colleagues [147] assessed the invaders affecting native ichthyofaunas severely [161,162]. morphometric variability of the three most common Given the importance of inland fisheries and goramy strains reared in Java (i.e. Baster, Blusafir and traditional aquaculture in Indonesia for the economy and Paris) and found no diagnostic characters distinguishing food safety of the country, the Indonesian government those strains other than coloration patterns. Recently, declared its concern about invasive species by the Decree however, Nuryanto and colleagues [148] demonstrated by of Ministry of Agriculture No. 179/1982 dated March 22, sequencing the mitochondrial COI gene that five of several 1982. Early in the 80’s, a blacklist of exotic species to strains in central Java (i.e. Blue Saphire, Jepang, Mutiara, be controlled at Indonesian borders was established Sabah and Sowang) were build upon two distinct species (Serrasalmus spp., Vandellia spp., Lepisosteus spp., while actual O. goramy strains show little differentiation. Silurus glanis, Esox masquinongy, Electrophorus electricus These preliminary results highlight that the history of and Tetraodon spp.), mainly focusing on potentially the domestication of the giant goramy in Sundaland harmful species for human populations and thereby, has been complex, involving different species during threatening human activities related to the harvesting of the development of current strains and a large scale freshwater fishes. This decree, however, did not succeeded reappraisal of their biology and evolutionary relationships in impeding the introduction of several of those species are urgently needed to promote a sustainable valorization including the alligator gar (Lepisosteus sp.) that has been of those strains. recently reported from Java and Indonesian Papua (R. The catfish belonging to the genera Pangasius and Hadiaty, pers. obs.). Furthermore, little attention has been Clarias (Fig. 5) are other iconic species of the freshwater paid to other species well known for their high invasive aquaculture production and fisheries in Indonesia but potential such as the Tilapias (Oreochromis niloticus and also more broadly in SEA. Despite the overexploitation of O. mossambicus), the carp (Cyprinus carpio), the guppy their natural populations and rearing in captivity for food (Poecilia reticulata), the North African catfish (Clarias consumption, the taxonomy of the genus Pangasius have gariepinus) or the Amazon sailfin catfish (Pterygoplichthys been subject to various changes during the last two decades spp.) that have been introduced for aquaculture or [11]. In 1991, Roberts and Vidthayanon [149] revised the escaped through the ornamental trade and are now widely family Pangasiidae and reported 21 valid species based on established in Indonesia, sometimes dominating the anatomical and morphological characters. Later, Pouyaud biomass of the fish communities (R. Hadiaty, pers. obs.). and colleagues reappraised the taxonomy and species More surprisingly, asiatic species have been introduced richness of the family in Indonesia based on sequencing of for aquaculture purposes while several candidates species mitochondrial DNA in combination with morphology and belonging to the same genus were readily available in described five additional species [150-154]. Two species of the native Indonesian ichthyofauna. The North African Pangasiidae are reared in Indonesia [155], Pangasianodon catfish (Clarias gariepinus), for instance, is an emblematic hypophthalmus and Pangasius djambal, the former species of the aquaculture trade that is now cultivated in

Unauthenticated Download Date | 12/4/15 10:41 PM 158 N. Hubert, et al. several countries worldwide and has become one of the for the “unrecognized diversity“ reported by Collins and most popular cultivated species in Java (i.e. Lele jumbo), colleagues [170]. Collins and colleagues [170] highlighted for example. The genus Clarias, however, is a speciose that mismatch between species names applied to the catfish genus that has been through important taxonomic specimens transiting in the international ornamental revisions recently, leading to the description of 8 new trade and taxonomic assignment based on DNA barcodes species [163-169] and raising the number of Clarias species are due to either taxonomic uncertainty and/or conflict to 13. Worth mentioning, 11 of those 13 species are endemic due to misidentifications. The authors hence caution the of Sundaland and escapes of C. gariepinus from cultivated use of GenBank for identification purposes due to the ponds are expected to have impacted their abundances potential absence of preserved voucher specimens and in nature through competitive interactions and epizooty. justified identifications and note that while BOLD data are Border security officers, however, often lack taxonomic generally better curated and with higher quality standards, training needed to identify potential invasive species they are also likely to suffer from misidentification to some or species known to carry exotic pathogens. Collins and degree. This case highlights the difficulties of establishing colleagues [170], for instance, explored the usefulness of DNA barcode reference libraries when the taxonomic DNA barcodes in identifying ornamental fishes obtained knowledge is rapidly evolving as exemplified with the from the aquarium trade. They focused on cyprinids Indonesian ichthyofauna. and included several popular aquarium fishes that are The freshwaters of Sulawesi are, due to the island’s regularly exported from Indonesia, mostly Sumatra location east of Wallace’s line, devoid of those freshwater and Kalimantan. A total of 678 specimens, representing fish lineages making up the vast species diversity of 45 genera and 172 cyprinid species were obtained by the islands formerly connected to Sundaland [10]. In the authors from the aquarium trade. In addition, 562 contrast, Sulawesi’s inland waters, and particularly the barcode sequences from 238 species were obtained from ancient lakes, are dominated by species flocks originating reference libraries (GenBank/BOLD) increasing the total from a few lineages, such as ricefishes (Oryzias, number of ornamental cyprinid species included in the Adrianichthys), the sailfin silversides (Telmatherina, study to 329. Many morphological similar species were Paratherina, Tominanga) mentioned above, halfbeaks well differentiated with DNA barcodes and identification (Nomorhamphus, Dermogenys), and gobies (including success rates were generally high. The authors also flocks of Glossogobius and Mugilogobius) but rivers and observed, however, incongruence and inconsistencies streams of the island are dominated by euryhyaline in the DNA barcode data. For example, barcode sharing species [10,172]. Alien fish species such as the snakehead was observed in several groups, including taxa from Channa striata, the climbing perch Anabas testudineus, Indonesia: between two Eirmotus species (E. cf. insignis the gouramis Trichopodus pectoralis and T. trichopterus, and E. cf. octozona) and between two Boraras species (as or Tilapia (Oreochromis niloticus) are omnipresent in many Rasbora in (Collins et al. 2012), B. brigittae and B. merah). areas of Sulawesi, but actual data on their occurrence or In the case of Boraras, Kottelat [11] commented on the even impact to the native fauna are very restricted, and taxonomic status of B. merah mentioning the potential scattered in the literature. color dichromatism in this species, but also highlighting A recent checklist of alien fish species reported 14 the clear differences between the two species (shape species occurring, and partially spreading heavily, in the of caudal peduncle, pigment distribution). Hence, it is Malili Lakes, Sulawesi’s largest lakes system [173]. These likely that the supposed barcode sharing between the two include species commonly used for aquaculture on a local species is due to misidentifying B. merah as B. brigittae. scale, such as snakeheads, gouramis, climbing perches, The results of Collins and colleagues [170] also or walking (Clarias) from Western Indonesia or indicated a high level of unrecognized diversity. Among mainland Asia, but also African Tilapias, aquarium species Indonesian taxa, for example, unrecognized diversity such as the Amazon sailfin catfish (Pterygoplichthys cf. was found in Brevibora dorsiocellata (as Rasbora in pardalis), guppy (Poecilia reticulata), African cichlids [170]), Rasbora einthovenii, Trigonostigma heteromorpha from Malawi lake (e.g. Pseudotropheus cyaneorhabdos), (as Rasbora in [170]), Trigonopoma pauciperforata (as or common carp (Cyprinus carpio). Most of these species Rasbora in [170], a widespread species) and Sundadanio apparently do not pose a direct threat to the native lake axelrodi. At the same time, Brevibora cheeya (Liao & Tan, species, but there is at least one exception. The artificial 2011) from Terengganu, Malaysia was described and hybrid, the flowerhorn cichlid (also called Lou Han), bred recently Brevibora exilis [171] from Kalimantan Tengah, as colorful ornamental fish in captivity, entered Lake Borneo was described and both could potentially account Matano just a few years ago, but spread extremely fast

Unauthenticated Download Date | 12/4/15 10:41 PM DNA Barcoding Indonesian freshwater fishes: challenges and prospects 159 throughout the lake. Detailed studies on its impact are based on a revision of available sequence data and pending, but the restricted data available suggest that this newly generated sequences with the aim to enable is an omnivorous benthic feeder, potentially interacting identification of potential and established invasive with the native flocks by competition and predation species outside of their native range. A total of 49 [173]. The even more recent introduction of the Amazon discrete haplogroups were identified and multiple sailfin catfish highlights the multitude of artificial – and divergent clusters were observed within four Channa unnecessary – introductions, each of which is a large- species including two species that also occur in scale experiment at the cost of Sulawesi’s precious Indonesia (C. gachua and C. striata) suggesting the ancient lakes fauna. DNA barcoding, applied to specimen potential presence of cryptic species diversity within samples or as “environmental barcoding” to water probes, these lineages. This knowledge is of prime importance, has the potential to enhance the recognition of alien however, for monitoring snakehead species invasion as species in inland waters, once the reference libraries are the family exhibits a high diversity of life history traits, made available; monitoring the spread and impact of non- particularly temperature tolerance that influences native fish species in Indonesia’s lakes, rivers and streams species invasive potentials [176]. appears timely. Alternatively, SEA is an important source for exports internationally and several introductions of SEA fishes 5 DNA barcoding Indonesian fresh- in temperate zones, North America for instance, with water fishes: perspectives adverse effect on native ichthyofauna have been recently reported such as the striped snakehead [174]. In fact, 5.1 DNA barcode reference libraries: toward several snakehead species (Channa spp.) have been introduced in non-native waters around the world where a global biodiversity information system for many developed into invasive species threatening the species identifications indigenous ichthyofauna. Channa striata is considered the DNA barcoding is a system designed to provide accurate, most widely introduced species of snakehead. Although fast and automatable species identification by using short it is generally accepted that the native range of C. striata and standardized gene regions as internal species tag includes Borneo, Roberts [175] suggested that its presence [14]. As such, DNA barcoding is at the crossroad, while in western Borneo might have been due to introductions. complementing, several areas of biodiversity sciences C. striata has been introduced throughout Indonesia in including taxonomy, phylogeny and populations genetics Sulawesi, Lesser Sundas, Moluccas and Papua [176]. There [180,181]. The initial proposal by Hebert and colleagues are often contradictory reports regarding the taxonomic [15], consisted in acknowledging the effectiveness of identity of some of the introduced snakehead, for example mitochondrial COI gene in capturing species boundaries the blotched snakehead (Channa maculata) that has been and highlighting that the discriminating power of COI gene established on Oahu, Hawaii, since the late 1800s has at the species level might be used for species identification often been misidentified as the striped snakehead (C. based on DNA sequences. DNA barcoding relies on the striata). development of DNA barcode reference libraries for The snakeheads (Channidae) comprise 34 species known species in order to foster automated assignment in two genera, Parachanna with 3 species in Africa and of unknown specimens to known species. For automation Channa with 31 species in Asia where their distribution purposes, both stability of the reference libraries and range from Iran in the West to the Amur basin in Russia reproducibility of the molecular identifications should and Sundaland in the south with 12 species (Appendix). be guaranteed [182]. For this purpose, the Barcode of Life Snakeheads are highly valued as food and are used in Datasystem (BOLD) hosts specimen records consisting of fisheries, aquaculture, and the life-food trade and can seven data elements (Fig. 6): be found in most fishmarkets in SEA. Their taxonomy 1. Species name and systematic has been controversial in the past, the 2. Voucher data first molecular frameworks for channid phylogenetics 3. Collection record were published based on mitochondrial genes [177] 4. Identifier of the specimen or combining mitochondrial and nuclear genes [178]. 5. COI sequence of at least 500 bp More recently, Serrao and colleagues [179] established 6. PCR primers used to generate the amplicon a Channidae DNA barcode reference library based on 7. Trace files 250 DNA barcodes sequences representing 25 species

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These detailed specimens and barcode records ensure the 5. Links to voucher specimens using the approved format reproducibility of the PCR and sequencing protocols by of institutional acronym:collection code:catalog ID providing contact of the peoples involved in generating DNA number. barcodes. In addition, it enables the continuous updating Altogether, these data allow connecting voucher of the identification of specimens by the community of specimens, further available for screening diagnostic users and facilitates the use of DNA barcodes for further morphological characters anytime undescribed diversity taxonomic studies [21,27-31,183]. To further improve the is detected, with DNA barcodes. quality of DNA barcode data, the BARCODE data standard as been implemented in GenBank for the records that are 5.2 DNA barcoding: a solution to the compliant with the following requirements: taxonomic impediment? 1. Bi-directional sequences of at least 500 base-pairs from the approved barcode region of COI, containing no The broad development of DNA-based assessment of ambiguous sites biodiversity opened new perspectives in the inventory 2. Links to electropherogram trace files available in the of earth living beings [180]. In particular, it has been NCBI Trace Archive highlighted through the use of DNA-based approach of 3. Sequences for the forward and reverse PCR amplification species inventories that describing biological diversity primers with traditional approaches happens at a much slower 4. Species names that refer to documented names in a rate than the rate of species loss [18]. Compared to taxonomic publication or other documentation of the morphological characters, DNA barcodes presents several species concept used advantages: (1) intraspecific phenotypic variation often

Figure 6. Structure of a specimen record in BOLD. The BARCODE keyword in GenBank is reserved for the records compliant with the following scheme including a voucher specimen in a biological collection, a tissue sample in a bio-repository, collection data, a specimen photograph and a DNA barcode including primary data (e.g. trace files).

Unauthenticated Download Date | 12/4/15 10:41 PM DNA Barcoding Indonesian freshwater fishes: challenges and prospects 161 overlaps that of sister taxa in nature, which can lead to to increase the number of species descriptions based on incorrect identifications or species delineations [22], such iterative procedures. (2) DNA barcodes are effective whatever the life stages under scrutiny [24,184]. Recent large-scale DNA barcoding 5.3 New tools for species identification, campaigns have demonstrated that DNA barcoding will environmental DNA barcoding and the have to face the undescribed diversity of earth biotas management of ichthyological resources [e.g. 27-29] and that DNA barcodes might help to speed up the pace of species discovery through automated Recent advances in molecular biology have opened up delineation of mitochondrial lineages. This approach is new perspectives for the management of ichthyological particularly relevant when applied to areas of exceptional resources, e.g. the possibility to record rare and threatened diversity facing massive anthropogenic threats and as species, to describe the species composition or to elucidate such, urgently awaiting more comprehensive assessments ecosystem processes from a broad range of environmental [1-3,185]. samples [190-193]. In contrast with DNA extracted from Recently, the effectiveness of such iterative procedures well-preserved organisms, environmental DNA (eDNA) including DNA barcoding, taxonomy and natural history consists of a mixture of potentially degraded DNA present in the delineation of species has been highlighted in in trace amounts from many different organisms [190]. speciose communities [27,186-188]. Procedures including Since the long size of the “universal” COI barcode target DNA barcoding as a first step toward the fast description (658 bp long) is not suitable for a successful amplification of species based on the combination of COI sequences, of degraded DNA, some authors have introduced a concise morphological descriptions, and high-resolution universal “mini-barcode” of 130 bp [194-197] that proved digital imaging enable the description of larger number to be particularly effective for the DNA barcoding of old of species. In addition, the automated and objective museum specimens. This new approach of monitoring classification of mitochondrial lineages enabled by the biodiversity already proved to be effective in a variety the use of DNA barcode reference libraries provides of field including functional ecology [e.g. 196,198], a potential solution to the time consuming sorting of biodiversity assessment and environmental inventorying specimens during inventories of speciose, yet unknown [e.g. 192,199], re-appraisal of archive specimens in natural fauna [187,188]. This procedure enables to speed up the history museums [e.g. 186,197] and monitoring of the taxonomic workflow through a preliminary sorting of ornamental trade [e.g. 170]. specimens according to their DNA barcodes followed by an This mini-barcode approach was also recently iterative procedure involving natural history, morphology implemented in a biosecurity perspective for freshwater and DNA barcoding [e.g. 27,189]. fish species transiting in the international ornamental Studies based on similar iterative procedures and trade using eDNA from water of quarantine tanks [170]. In molecular data compliant with DNA barcode data their study, the authors targeted a single species (Danio standard, however, have been scarcely applied during the rerio) and COI was successfully detected at fish densities inventory of the Indonesian ichthyofauna. Kadarusman as low as 0.08 g/L, further illustrating the potential of this and colleagues [13,90] pioneered the application of approach as a tool for monitoring quarantine facilities for this approach in the Indonesian ichthyofauna and a variety of invasive and/or endangered species, in the exemplified its benefits by the simultaneous description context of the international ornamental fish trade. Here, of eight new species of rainbowfishes from the Papua’s authors defined a mini-barcode for the species-specific bird head based on an initial screening of species detection of Danio rerio with the use of a sliding window diversity through DNA barcodes. Later, Larson and function from the DNA barcoding package Spider [200]. colleagues [85] adopted the same iterative procedure for In addition, short-length DNA barcodes have the the Mugilobius of Sulawesi and described a new species. advantage of better fitting the sequence length of most Along the same line, Parenti and colleagues successfully of the next-generation sequencing (NGS) platforms. NGS implemented a similar procedure for the Oryzias spp. is indeed increasingly used to analyze the entire species of Sulawesi and described two new endemic species composition either from pools of entire organisms or characterized by both private mitochondrial lineages from environmental samples. This high-throughput and diagnostic morphological characters [84]. Such multispecies identification approach was recently studies have been scarce so far during the ichthyological designated under the term “DNA metabarcoding” [201]. exploration of the Indonesian inland freshwaters but the Proofs of concept of this so-called DNA metabarcoding ease of access to sequencing facilities may be expected have been successfully achieved and have shown its

Unauthenticated Download Date | 12/4/15 10:41 PM 162 N. Hubert, et al. great potential for deciphering the species richness associated data standard will play an increasing role in and composition from a wide variety of samples [191- the future. 193,198,202]. Addressing the potential of environmental DNA for monitoring rare and threatened freshwater species, Thomsen and colleagues [203] demonstrated that 6 Conclusions the diversity, and even the abundance, of such species The present review highlights the progress achieved could be monitored from DNA recovered from water so far on the taxonomic knowledge of the Indonesian samples of ponds, lakes and streams. They also showed ichthyofauna. Nearly 1200 native species have been either that DNA could be detected up to two weeks after reported or described from Indonesian freshwaters but had been removed from the analyzed water. the disrupted flow of taxonomic works during the last Though these new approaches look very promising centuries and the fast acceleration of the rate of species for the management of Indonesia ichthyological resources description during the last decades highlights that much (which includes activities as diverse as the assessment remain to be done. The taxonomy and systematic of many of biodiversity, the early detection of invasive species, lineages is still pending a major revision and this gap is the monitoring of elusive species or the control of currently bridling the sustainable management of many quarantine facilities), yet many questions arise about the species of economic potential for either consumption or accuracy and reliability of environmental DNA barcoding. the ornamental trade. Considering the fast intensification The primary source of uncertainty comes from the of the anthropogenic perturbation in Indonesia, filling manipulation and processing of eDNA, which require a this gap has become critically important for conservation rigorous standardization and quality control assessment. purposes. DNA barcoding has the potential to help This seems to be widely acknowledged and solutions to increase rapidly this knowledge and to help promote more limit these biases have been proposed [see 190,204]. The sustainable practices in taxonomy through the linkage second source of error lies in the reproducibility of NGS between traditional and molecular taxonomy with online methods, which has not been thoroughly evaluated in the data systems and the development of new molecular frame of biodiversity assessments. One of the rare studies tools for species identification. The main challenge focusing on this aspect highlighted the necessity to run at the moment is the development of comprehensive replicates in order to reduce the risk of misestimating DNA barcodes reference libraries for the Indonesian species diversity, particularly in regard to rare taxa [199]. ichthyofauna. Given the complex systematic and Finally, the accuracy of the DNA-based identifications is taxonomy of many freshwater fish lineages at the moment, tightly dependent on the coverage of the DNA barcode this review and its associated database in Appendix is reference libraries used to assign the sequences generated expected to help streamlining future campaigns of DNA through NGS to known taxa [e.g. 17,21,28,189,205-209]. barcoding in the country. Whatever the type of sample used, the taxonomic identification of species relies on the ability to match Acknowledgements: The authors wish to thank Dr. Siti sequences with the DNA barcodes reference librairies. Nuramaliati Prijono, Dr. Tjak Witjaksono, Mohammad While mini-barcodes are compatible with DNA barcodes Irham, Dr. Marlina Adriyani, Dr. Rosichon Ubaidillah, data standard (i.e. voucher specimens deposited in formal at Research Centre for Biology (RCB-LIPI), Dr. Jean-Paul collections, accessibility to collection metadata) and Toutain, Dr. Jean-François Agnčse and Dr. Domenico Caruso match the standardized DNA barcode system [181,182,210], from the ‘Institut de Recherche pour le Développement’, and yet another major limitation for the implementation of Dr. Bambang Suryobroto at the Bogor Agronomy University DNA metabarcoding in the management of the Indonesian for their support. We are thankful to Dr. Daisy Wowor at ichthyodiversity is the necessity to expand the current RCB-LIPI, Sumanta and Bambang Dwisusilo at IRD coverage of those libraries for freshwater fishes beyond for their help. The authors thank the anonymous referees those currently available. Worth mentioning, NGS opened for their helpful comments. This publication has ISEM new perspectives in the application of routine DNA-based number 2015-180 SUD. species identification and suggested that DNA sequencing may eliminate the required DNA amplification step [201]. Conflict of interest: Authors declare nothing to disclose. If so, the DNA barcoding reference libraries and their

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